Method for dissociating biotin complexes

ABSTRACT

A method for dissociating a complex of a biotin compound and a biotin-binding compound, by contacting the complex with an amine, is disclosed.

BACKGROUND OF THE INVENTION

Today many powerful techniques, derived from research in molecularbiology, are ready to be used in routine diagnostic or forensicapplications. Prominent examples are the polymerase chain reaction, PCR(Saiki, R. K. et al. (1985) Science, 230, 1350-1355), based on a cyclic,template-directed primer extension reaction; the analysis of restrictionfragment length polymorphisms (Kan, Y. W. and A. M. Dozy (1978), Lancet2, 910-912); and the ligase chain reaction (Barany, F. (1991)Proc.Natl.Acad.Sci.USA, 88, 189-193) to detect known point mutations atthe ligation site of adjacent oligonucleotides.

It appears likely that in the near future, application of thesetechniques for analysis of DNA will augment or supplant conventionaldiagnostic procedures based, for example, on the detection ofdisease-associated metabolites.

Currently, the most common tool for the analysis of DNA is fragmentseparation by gel electrophoresis. However, in many cases,electrophoretic analysis and subsequent detection of labeled fragmentsis more time-consuming than performing the enzymatic reaction, andtherefore is a time-limiting step.

Detection techniques are under development which will enhance signalacquisition and provide automated and parallel sample processing, andwill likely lead to cost-efficient and time-saving sample processing indiagnostic and forensic applications. Also, large DNA sequencingprojects, such as the Human Genome Initiative, that seek to sequencegenes or entire genomes, for research or diagnostic purposes, requireautomated techniques with a high throughput to ensure timely completionof the project.

A promising tool which meets at least some of these criteria is theanalysis of DNA fragments by matrix assisted laser desorption/ionisationtime-of-flight (MALDI-TOF) mass spectrometry (Karas, M. and Hillenkamp,F. (1988) Anal. Chem., 60, 2299-2301).

The biotin-streptavidin system is a common and useful tool for thepurification of biotinylated materials (X. Tong and L. M. Smith (1992)Anal. Chem., 64, 2672-2677), e.g., products from PCR or sequencingreactions. Streptavidin (and also avidin) are bacterial proteins whichform tight complexes with biotin, including biotin conjugated to othermolecules such as nucleic acids. The stability of thebiotin-streptavidin complex during intensive washing permits removal ofnon-specifically bound and non-biotinylated material, which is of greatimportance for the success of reaction product analysis. The propertiesof the biotin-streptavidin complex can be used in systems employingbiotin bound to streptavidin on a solid support to yield immobilized ofbiotinylated molecules. The solid phase, including the complexedbiotinylated molecules, can be physically collected for furthermanipulations, including i) removal of excess reaction components likebuffer salts, enzymes or deoxynucleotide triphosphates (dNTPs) or ii)performance of enzymatic reactions like nucleolytic digests and solidphase sequencing.

A broad spectrum of applications for the biotin-streptavidin system isknown and even techniques not yet developed will be adaptable to thissystem (see, e.g., Stahl et al. Nucleic Acids Research (1988) 16,3025-3038; Hultman et al. Nucleic Acids Res. (1989) 17, 4937-4946;Hornes et al. Genet. Anal. (1990) 7, 145-150).

Although the biotin-streptavidin complex is the result of non-covalentbonding, the affinity of streptavidin for biotin is about one milliontimes more powerful than that of most antibody-antigen interactions.However, for the analysis of reaction products it is important toprovide conditions for an effective dissociation of the complex whilerecovering the analyte molecules without modification.

Currently the recovery of biotinylated substances is based onbiotin-streptavidin complex dissociation using substances like phenol,urea, or, most preferably, 95% formamide at temperatures between 25 and100° C. (Cocuzza et al., U.S. Pat. No. 5,484,701, 1996).

However, the use of formamide has been shown to be harmful for samplecrystallization, a necessary process for MALDI-TOF analysis, and forvarious enzymatic reactions (e.g. reactions employing alkalinephosphatase). Therefore methods based on the use of formamide are onlyuseful for subsequent gel electrophoretic analysis, but are harmful ifenzymatic reactions should be performed involving the isolated materialor other analytical tools are to be applied. The endo- or exonucleolyticdigest, for example, of PCR products would benefit from a methodallowing isolation of single-stranded PCR products which can be digestedafter purification.

Gel electrophoresis, the time and sample throughput-limiting factor inDNA diagnostics, will be replaced by more efficient techniques in thenear future. These applications suggest, that efficient methods linkingthe biotin-streptavidin technology to for example, MALDI-TOF MS arestrongly required.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for dissociating acomplex comprising a biotin compound and a biotin-binding compound. Themethod includes contacting the complex with an effective amount of anamine, under conditions such that the complex is dissociated, therebyforming a biotin compound and a biotin-binding compound.

In preferred embodiments, the complex is contacted with an amine at atemperature of between about 25° C. and about 100° C. In preferredembodiments, the biotin compound is a biotinylated macromolecule. Inpreferred embodiments, the biotinylated macromolecule is selected fromthe group consisting of a biotinylated nucleic acid sequence, abiotinylated protein, a biotinylated carbohydrate, and a biotinylatedlipid. In preferred embodiments, the biotin-binding compound is selectedfrom the group consisting of avidin, streptavidin, and derivativesthereof.

In preferred embodiments, the biotin-binding compound is immobilized ona solid support. In preferred embodiments, the solid support is amagnetic bead.

In certain preferred embodiments, after the contacting step, the biotincompound is separated from the biotin-binding compound. In preferredembodiments, after the separation step, the biotin compound is purified.In preferred embodiments, the biotin compound is purified by a methodselected from the group consisting of lyophilization, precipitation,filtration, and dialysis.

In preferred embodiments, prior to the contacting step, the complex ispurified.

In other preferred embodiments, the biotin compound is immobilized on asolid support.

In preferred embodiments, after dissociation of the complex, at leastone of the biotin compound and the biotin-binding compound is analyzedby mass spectrometry.

In certain preferred embodiments, after dissociation of the complex, thebiotin-binding compound retains biotin-binding activity. In preferredembodiments, after dissociation of the complex, the biotin moiety of thebiotin compound remains substantially intact.

In particularly preferred embodiments, the amine is ammonia. In otherpreferred embodiments, the amine is a primary amine.

In another aspect, the invention provides a method for analyzing abiotinylated nucleic acid. The method includes the steps of contactingthe biotinylated nucleic acid with a biotin-binding compound, therebyforming a biotinylated nucleic acid:biotin-binding compound complex andcontacting the complex with an effective amount of an amine, underconditions such that the complex is dissociated, thereby releasing abiotinylated nucleic acid and a biotin-binding compound; and analyzingthe biotinylated nucleic acid.

In preferred embodiments, the nucleic acid is DNA. In preferredembodiments, the biotin-binding compound is immobilized. In preferredembodiments, the biotinylated nucleic acid is analyzed by massspectrometry. In preferred embodiments, the amine is ammonia.

Thus, in one embodiment, the subject method provides a process forisolating biotinylated single-stranded or double-stranded DNA fromenzymatic reactions for the purpose of purification and sampleconditioning, which can be followed by subsequent analysis (e.g., bymass spectrometry) or further enzymatic reactions.

The above and further features and advantages of the instant inventionwill become clearer from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of the purification of biotinylated PCRproducts where the reaction was carried out in solution.

FIG. 2 is a schematic drawing of two different means for detectingSanger sequencing products.

FIG. 3 is a schematic drawing of different means for detecting PCRdigest products with single- or double-stranded specific endo- orexonucleases to generate DNA sequence information.

FIG. 4 shows a MALDI-TOF MS spectrum of a biotinylated 20 meroligodeoxynucleotide (SEQ ID NO: 1) immobilized on streptavidinDynabeads and recovered using the method as described in Example 1.

FIG. 5 shows a MALDI-TOF MS spectrum of a PCR product from the hepatitisB core antigen coding DNA region, purified with streptavidin Dynabeadsand recovered from the beads using ammonium hydroxide, as described inExample 2.

FIG. 6 shows the A-reaction of the Sanger sequencing reaction purifiedwith M-280 streptavidin Dynabeads.

FIG. 7 shows a mass spectrum of an exonucleolytic digest of a 60 mer PCRproduct.

FIG. 8 shows an exonucleolytic digestion of a biotinylated 25 merimmobilized on Dynabeads.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention features a method for dissociating biotincompounds, including biotin-conjugated (biotinylated) carbohydrates,proteins, polypeptides, peptides and nucleic acid molecules (e.g.,double-stranded or single-stranded DNA or RNA), from biotin-bindingcompounds, including streptavidin or avidin compounds. Once isolated,the biotin compound (or biotin-binding compound) can be analyzed usingvarious detection methods and/or employed in further enzymaticreactions.

The term "biotin compound", as used herein, refers to biotin and biotinderivatives and analogs. Thus, "biotin compounds" include compounds suchas biotin, iminobiotin, and covalent or non-covalent adducts of biotinwith other moieties. Preferred biotin compounds retain the ability tobind to avidin or streptavidin, or other biotin-binding compounds. Forexample, biotin has been used to derivatize a variety of molecules,including both small molecules (for example, chelating agents, e.g., ¹⁸⁶Re-chelators conjugated to biotin, see, e.g., U.S. Pat. No. 5,283,342 toGustavson et al.) as well as large molecules, including biomolecules(e.g., nucleic acids (including DNA, RNA, DNA/RNA chimeric molecules,nucleic acid analogs, and peptide nucleic acids), proteins (includingenzymes and antibodies), carbohydrates, lipids, and the like). Methodsof conjugating biotin to other molecules ("biotinylation") are wellknown in the art, and a variety of biotinylating reagents arecommercially available (from, e.g., Pierce, Rockford, Ill.). A varietyof coupling or crosslinking agents such as protein A, carbodiimide,dimaleimide, dithio-bis-nitrobenzoic acid (DTNB),N-succinimidyl-S-acetyl-thioacetate (SATA), andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),6-hydrazinonicotimide (HYNIC), N₃ S and N₂ S₂ can be used in well-knownprocedures to synthesize biotin amide analogs or biotin compounds. Forexample, biotin can be conjugated via DTPA using the bicyclic anhydridemethod of Hnatowich et al. (Int. J. Appl. Radiat. Isotop. 33, 327(1982)). In addition, sulfosuccinimidyl 6-(biotinamido) hexanoate(NHS-LC-biotin (which can be purchased from Pierce)), "biocytin", alysine conjugate of biotin, can be useful for making biotin compoundsdue to the availability of a primary amine. In addition, correspondingbiotin acid chloride or acid precursors can be coupled with an aminoderivative by known methods. Thus, preparation of a variety of moietiesconjugated to a biotin compound is possible. Furthermore, a biotincompound can be conjugated to a solid support, if desired.

The term "conjugated," as used herein, means ionically or covalentlylinked or attached (e.g., by use of a derivatizing reagent).

The term "biotin-binding compound" refers to a compound which cantightly but non-covalently bond biotin compounds. Biotin-bindingcompounds include avidin and streptavidin, as well as derivatives andanalogs thereof, including avidin or streptavidin conjugated to othermoieties (such as other proteins). Preferred biotin-binding compoundsinclude avidin, streptavidin, covalent adducts of avidin andstreptavidin, and fusion proteins having a domain which hasbiotin-binding activity. In certain embodiments, anti-biotin antibodiescan be used as the biotin-binding compound. The covalent linkage ofbiotin-binding compounds such as streptavidin is well known, and somestreptavidin-conjugated solid supports are commercially available (e.g.,Dynabeads magetic microbeads, Dynal, Hamburg, Germany). Thus,biotin-binding compounds linked to an insoluble support are useful inthe present invention.

A "solid support" refers to a support which is solid or can be separatedfrom a reaction mixture by filtration, precipitation, magneticseparation, or the like. Exemplary solid supports include beads (such asSepharose, Sephadex, polystyrene, polyacrylamide, cellulose, Teflon,glass, (including controlled pore glass), gold, or platinum); flatsupports such as membranes (e.g., of cellulose, nitrocellulose,polystyrene, polyester, polycarbonate, polyamide, nylon, glass fiber,polydivinylidene difluoride, and Teflon); glass plates, metal plates(including gold, platinum, silver, copper, and stainless steel); siliconwafers, mictrotiter plates, and the like. Flat solid supports can beprovided with pits, channels, filter bottoms, and the like, as is knownin the art.

A "biotin compound:biotin-binding compound complex" refers to anon-covalent complex formed by the binding of a biotin compound to abiotin-binding compound.

The term "analyzing," as used herein, refers to detection of, orcharacterization of, a molecule or moiety. Thus, a molecule can beanalyzed by a variety of known techniques, including spectrometrictechniques such as UV/VIS, IR, or NMR spectroscopy, mass spectrometry,chromatography, electrophoresis, or other methods known in the art, orcombinations thereof. "Analyzing" can also include methods such assequencing of nucleic acids.

The term "ammonia", as used herein, refers to NH₃, or any salt thereof.Thus, depending upon the solvent used, and the pH of the solvent,ammonia may be present as NH₃, or may be in the form of an ammonium saltor compound. For example, ammonia in aqueous solution can exist largelyas ammonium hydroxide (depending on the pH), but is generally referredto herein as "ammonia."

The term "amine", as used herein, refers to a compound having thestructure NR'₃, or N⁺ R'₄ in which R' is a hydrogen, alkyl (includingcycloalkyl), alkenyl, alkynyl, or aryl group, and can be independentlyselected for each occurrence. Two or more R' groups can be selected suchthat they form, together with the nitrogen atom to which they areattached, a cyclic amine (for example, pyrrolidine or piperidine). Incertain embodiments, aromatic amines such as pyridine are contemplatedfor use in the subject methods. Where an R' group is alkyl, the alkylgroup can have from one to twelve carbon atoms in a straight or branchedchain. Lower alkyls (having from one to six carbon atoms in a branchedor straight chain) are preferred. A preferred aryl group is phenyl,which can be substituted or unsubstituted. Exemplary amines includeammonia, methylamine, diethylamine, aniline, and diisopropylethylamine.Further, quaternary amines, such as tetrabutyl ammonium (e.g., astetrabutylammonium hydroxide), can be used in the methods of theinvention. Other nitrogen-containing compounds, including hydrazine, andderivatives and analogs thereof, can also be used in the invention.

It is believed that small amines (i.e., those molecules with smallsteric bulk, e.g., where at least one R' is hydrogen or a small alkylgroup such as methyl) are more effective than larger amines atdissociating biotin compound:biotin binding compound complexes. Thus,amines in which each R' is sterically small (e.g., hydrogen or a smallalkyl group such as methyl) are preferred. In particular, primary aminesare more preferred than secondary amines, which are in turn morepreferred than tertiary amines, which are in turn more preferred thanquaternary amines. In general, an amine will be selected according tofactors such as cost, efficacy, ease of handling, ease of purificationof the desired products, and the like. Ammonia is a most preferredamine, at least in part because ammonia effectively cleaves biotincomplexes and is inexpensive, readily handled as either the gas orammonium hydroxide in solution, and easily removed when reaction iscomplete (e.g., by lyophilization). As described in more detail below,the use of ammonia as the cleaving reagent permits facile purificationof biotin compounds by removal of excess ammonia under a vacuum. Thus,in certain embodiments, an amine that is sufficiently volatile to beremoved by lyophilization is preferred. However, other methods ofpurifying a biotin compound or biotin-binding compound (or a mixture ofboth) can be employed, including, for example, dialysis, gelelectrophoresis, capillary zone electrophoresis, affinitychromatography, crystallization, column chromatography (e.g., gel orionic exchange chromatography), HPLC, and the like.

The methods of the invention are particularly useful in purification ofbiotinylated compounds. For example, a biotinylated compound can readilybe separated from non-biotinylated compounds simply by contacting areaction mixture with immobilized biotin-binding compound, e.g.,covalently bound to a solid support, followed by separation and washingof the immobilized complex of the biotin compound with thebiotin-binding compound. The biotinylated molecule can then be isolatedby dissociation of the biotin:biotin-binding compound complex, followedby separation of the biotin compound from the solid support. Thepurified biotinylated compound can then be lyophilized, further purifiedby conventional techniques, or the like. The skilled artisan willappreciate that a biotin-binding compound can be purified by ananalogous process, e.g., by contacting a reaction mixture containing abiotin-binding compound with an immobilized biotin compound to form acomplex, followed by purification and subsequent dissociation of thecomplex.

In the case of biotinylated nucleic acids, the biotin-streptavidinsystem can be used for the isolation of either single- ordouble-stranded nucleic acids, e.g., DNA. A preferred application is theisolation of a double-stranded PCR product, the subsequent removal ofthe non-biotinylated strand and the downstream processing of the singlestrands (Mitchell, L. G., et al. (1994) "Advances in BiomagneticSeparation," Eaton Publishing, Natick, Mass., USA, p. 31-48). Currently,denaturing of double-stranded products on streptavidin Dynabeads ispreferably done using 0.1 M NaOH. Other applications include thepurification of PCR and LCR products as well as purification of DNAsequencing products (Jurinke, C., et al., (1996) Anal. Biochemistry,237-174-181).

The exact mode of action of ammonia and other amines on biotindecomplexation is not known. It is believed that the pH of the reactionsystem is important to effectively decomplex biotin and biotin-bindingcompounds within a reasonable time. However, it is known that biotincomplexes are stable up to relatively high pH in the absence of ammonia.In particular, 0.1N sodium hydroxide solution (at a pH of about 13) isless effective at cleaving biotin compound:biotin-binding-compoundcomplexes than is 25% ammonium hydroxide solution. Ammonia is known tobe a strong hydrogen bond donor-acceptor. Without wishing to be bound byany theory, it is believed that the ammonia molecules are small enoughto diffuse into the biotin complex and disrupt the bonding (e.g.,hydrogen bonding) of the biotin moiety with a biotin-binding compound.The biotin compound retains the ability to bind to a biotin-bindingcompound after treatment with ammonia, and the biotin moiety can remainsubstantially intact.

Thus, the pH of the reaction mixture can affect the rate of complexdissociation. In preferred embodiments, the pH of the reaction mixtureis in the range from about 7.0 to about 14.0, more preferably from about8.0 to about 13.0.

The concentration of the ammonia or other amine is also important. Apreferred concentration is at least about 5% ammonia or amine, morepreferably about 10%, and still more preferably at least about 15%(w/v). Higher concentrations will generally result in more rapid complexdissociation. Accordingly, a concentration of about 25-28% is preferred.An ammonia concentration of 25-28% can be readily achieved withcommercially available ammonium hydroxide solutions.

The temperature at which the cleavage reaction is performed also affectsthe rate of complex dissociation. The temperature will be chosenaccording to considerations such as the rate of reaction (which will befaster at higher temperatures) and the stability of the components(e.g., the biotin compound and biotin-binding compound), which willgenerally be less stable at higher temperatures. In preferredembodiments, the temperature is in the range from about 25° C. to about100° C., more preferably from about 40° C. to about 80° C. A preferredtemperature is about 60° C. In certain embodiments, it may be preferredto perform the cleavage reaction under pressure (e.g., in the range of1-200 atm), or in a sealed reaction vessel (such as a sealed tube orbomb).

The reaction mixture can be an aqueous mixture such as a solution orsuspension, or a non-aqueous solvent can be employed, including, e.g.,methanol, ethanol, acetonitrile, dimethylformamide, and the like.Mixtures of solvents can also be employed (e.g., water/methanol orwater/acetonitrile). The solvent will in general be selected to becompatible with at least one of the biotin compound, the biotin-bindingcompound, or the amine. The choice of an appropriate solvent will beroutine to the skilled artisan. Aqueous solvents are generallypreferred.

Of course, the skilled artisan will appreciate that not all biotincompounds (or biotin-binding compounds) will be stable to all reactionconditions. For example, where a biotin compound is a biotinylatedprotein (e.g., a biotinylated antibody), vigorous conditions such ashigh pH and high temperature can denature the protein moiety. Thus,conditions will in general be selected to avoid undesired denaturationor destruction of at least one of the biotin compound or biotin-bindingcompound.

The method of the invention can be used to purify a biotin compound. Forexample, in a preferred embodiment, the method comprises the steps ofcontacting a biotin compound:biotin-binding compound complex with aneffective amount of ammonia, under conditions such that the complex isdissociated, thereby forming a biotin compound and a biotin-bindingcompound. In certain preferred embodiments, the method includes, priorto the dissociating step, the step of purifying the biotincompound:biotin-binding compound complex by separating the complex fromat least one impurity. Thus, by contacting a reaction mixture comprisinga biotin compound with a solid support comprising an immobilizedbiotin-binding compound, followed by purification of the complex andsubsequent release of the biotin compound from the complex by treatmentwith an amine, the biotin compound can be purified from a complexreaction mixture. Examples of the purification of, e.g., nucleic acids,by the methods of the invention are provided below. It has been foundthat the inventive methods are particularly useful where a biotincompound is to be analyzed by mass spectrometry subsequent to complexdissociation. In this embodiment, use of ammonia as the amine ispreferred.

Thus, in a preferred embodiment, the invention provides a simple methodfor isolating biotin-conjugated molecules from biotin-streptavidincomplexes. The method is compatible with subsequent mass spectrometricanalysis of the isolated biotin-conjugated molecules.

The subject method provides a mass spectrometric-compatible process forreleasing intact and non-modified biotinylated molecules frombiotin-streptavidin complexes by a brief treatment of a complex with anamine (e.g., ammonia, which can be supplied as ammonium hydroxide),preferably at slightly elevated temperatures. Several mass spectrometerformats can be used for detection of the recovered products, includingionization by matrix-assisted laser desorption/ionization (MALDI),continuous or pulsed electrospray (ES), or massive cluster impact (MCI);and detection formats including linear or reflectron time-of-flight(TOF), single or multiple quadrupole, single or multiple magneticsector, Fourier Transform ion cyclotron resonance (ICR), ion trap, andcombinations thereof. For ionization, numerous matrix/wavelengthcombinations (MALDI) or solvent combiantions (ESI) can be employed.

This new method is of outstanding importance for all processes which arebased on a fast and quantitative recovery of biotin-conjugated materialsfrom biotin-streptavidin complexes and subsequent enzymatic reactionsand analysis via techniques negatively influenced by organic impuritiessuch as formamide. These include, for example, analysis of PCR or DNAsequencing products with mass spectrometry for diagnostic purposes.

FIG. 1 illustrates one embodiment of the invention. FIG. 1 is aschematic drawing of the purification of biotinylated PCR products wherethe reaction was carried out in solution. In this scheme, A representsthe biotinylated PCR product, R represents reaction components andimpurities and H represents the streptavidin coated solid support (e.g.streptavidin Dynabeads or multititer plates).

In step 1 of this embodiment, a PCR reaction is carried out with atleast one biotinylated primer. The biotin group can be attached eitheron the 5'-hydroxyl group of the primer or at an internal base. Bychoosing appropriate conditions, as are known in the art, in thepresence of buffer, template, deoxynucleotides and a thermostable DNApolymerase (collectively depicted as R) a biotinylated PCR product (A)will be generated.

Since short primers will immobilize more efficiently than longer PCRproducts, in step 2 of FIG. 1, the non-extended primer is removed byultrafiltration through size-exclusion membranes, according toprocedures described in the art. After ultrafiltration, the PCR productwill sometimes be accompanied by enzyme and/or some buffer components(R). For further purification, the PCR product can be complexed to asolid support (H) having a biotin-binding compound immobilized thereon(e.g., streptavidin or avidin). The complexation conditions may vary;suitable conditions include incubation at ambient temperature in thepresence of 2M sodium chloride or ammonium chloride, and a pH of around7.5. The nature of the solid support may vary, and include, e.g.,magnetic particles (e.g., beads), multititer plates with or withoutfilter plates, glass, silicon wafers with or without pits, plastic,paper, flat arrays, capillaries, agarose or sepharose.Streptavidin-coated magnetic beads (Dynabeads, Dynal, Inc) arepreferred.

In step 3 of FIG. 1, due to the stability of the biotin-streptavidincomplex, intensive washing can be performed to remove all excessivereaction components prior to the recovery of the PCR product. Thecomplexed and purified PCR product is then treated with a small volumeof 25% ammonium hydroxide, preferably at about 50-60° C., to overcomethe biotin-streptavidin interaction.

In step 4 of FIG. 1, the pure PCR product is accompanied only byammonium hydroxide. The ammonium hydroxide can easily be removed bylyophilization or ethanol precipitation, both methods well known in theart. The pure PCR product is redissolved, most preferably in ultrapurewater. Other conditions can be chosen, for example varying buffers as asolvent. The PCR product now can be employed in downstream applications,e.g., enzymatic reactions or detection, for example, via massspectrometry or gel electrophoresis in slab gels or capillaries, asdescribed in the art.

Using ammonia for the dissociation of the biotin-streptavidin complex isof special interest for detection of DNA using mass spectrometry. It isknown that heterogenity of cations leads to a broadening of mass signaland interferes with the detection process. Thus, mass spectrometricdetection would benefit if cation homogeneity could be achieved. Themethods of the invention provide DNA with a homogeneous cationdistribution, because after treatment with ammonia, most of thephosphate groups will carry an ammonium counterion. Another advantage isthat DNA with ammonium counterions is known to have preferred propertiesfor mass spectrometric (e.g. MALDI-TOF) MS analysis.

A second embodiment of the subject method is illustrated in FIG. 2. Itfeatures the detection of Sanger sequencing products and the use of abiotin-streptavidin complex in the process. FIG. 2 is a schematicdrawing of two different means for detecting Sanger sequencing products.On the left side of this scheme, a biotinylated primer is used and thereaction is carried out in solution with subsequent immobilization,purification, ammonium hydroxide treatment and detection. On the rightside, the reaction is performed as a solid phase reaction. In thisscheme B represents biotinylated primer extension products, R representsreaction components and impurities, C represents template DNA, and Hrepresents the streptavidin-coated solid support.

As shown in FIG. 2, left column, in the first step, a Sanger sequencingreaction is carried out in solution with a biotinylated primer. Theproducts arising from this primer extension reaction are double-strandednucleic acids consisting of the extended biotinylated primer (B) and thetemplate strand (C). In the second step, as show in FIG. 2, thebiotinylated complex is immobilized on a streptavidin-coated solidsupport (H) (as described above). Due to the stability of thebiotin-streptavidin complex, the immobilized complex can be separatedfrom excess reaction components, by-products and impurities (R) aspreviously described. In the third step the template DNA is denaturedfrom the biotinylated primer extension products (B) by methods wellknown in the art. A solution of 8 M urea was used for the purpose ofdenaturation. Further washing steps, preferably with ultrapure water,were employed to remove the urea and the template strand. In the fourthstep the purified biotinylated sequencing product (B) was recoveredthrough treatment with 25% ammonium hydroxide. Removal of ammoniumhydroxide can be performed using ethanol precipitation orlyophilization, as described above. The biotinylated products can beresuspended in ultrapure water and analyzed by mass spectrometry or gelelectrophoresis or subjected to further enzymatic reactions.

In a variation of this second embodiment, the sequencing reaction iscarried out on a solid support. FIG. 2, right column, illustrates thisvariation. In the first step, the biotinylated primer is immobilized ona streptavidin-coated solid support (H). In the second step, a templatestrand is annealed to the immobilized biotinylated primer. In the thirdstep a sequencing reaction, such as Sanger sequencing, is carried out.In the fourth step, reaction components and impurities (R) are removedthrough washing. The template is denatured from the biotinylatedextended primer using urea as described above, and further washing stepsare carried out. In step 5 the purified biotinylated extension products(B) are recovered through treatment with ammonium hydroxide as decribedabove, and subjected to analysis, for example, via mass spectrometry,electrophoresis or further enzymatic reactions.

A third embodiment of the instant invention is illustrated in FIG. 3.FIG. 3 is a schematic drawing of different means for detecting PCRdigest products with single- or double-stranded specific endo- orexonucleases to generate DNA sequence information. In this scheme, Arepresents biotinylated PCR product; D represents digested biotinylatedPCR product; E represents biotinylated single-stranded PCR digestproduct; F represents undigested biotinylated single-stranded PCR digestproduct; G represents digested biotinylated single-stranded PCR digestproduct; H represents a streptavidin coated solid support; and Rrepresents reaction components and impurities.

This embodiment features the sequencing of PCR products with endo- orexonucleases and the use of the -biotin-streptavidin complex in thisprocess. In the first step, as shown in FIG. 3, left column, a PCRreaction is carried out with one biotinylated primer. Prior to furtherreactions, unincorporated primers are removed using ultrafiltrationthrough a molecular weight cutoff membrane (as decribed above). In thesecond step, the biotinylated PCR product (A) is subjected to digestionwith double-strand specific enzymes. This can be carried out using, forexample, DNaseI, which nicks the double strand DNA statistically at eachphosphodiester bond. The reaction can be carried out in a way such thateach double strand arising from the PCR reaction is nicked oncestatistically (Low, C. M. L. et al. (1984) Nucleic Acids Res. 124865-4879). A second approach is the use of exonuclease III, whichdigests double-strand DNA from the 3' end of the molecule. A thirdapproach uses type II restriction-endonucleases to carry out RFLPs incombination with subsequent analysis, for example, via mass spectrometryor electrophoresis. In the third step, the biotinylated digestionproducts (D) of step 2 are immobilized on a streptavidin coated solidsupport (H) (as described above). In step 4, the immobilizedbiotinylated products are separated from reaction components andimpurities (R) (as described above) and the hybridized non-biotinylatedproducts are removed using a denaturing agent like urea (as describedabove for the second embodiment). The biotinylated products (E) arereleased from the solid support by treatment with ammonium hydroxide.After removal of the ammonium hydroxide using ethanol precipitation orlyophilization, the biotinylated products (E) can be resuspended in, forexample, ultrapure water and analyzed, for example, via massspectrometry or electrophoresis.

A variation of this embodiment features the sequencing of PCR productswith single-strand-specific endo- or exonucleases and the use of abiotin-streptavidin complex in the process. It is illustrated in FIG. 3("Variation 1"). The biotinylated PCR product (A) is immobilized on astreptavidin-coated solid support (H) after ultrafiltration (step 2). Instep 3 the non-biotinylated strand is denatured using urea, and furtherwashing steps are carried out. In step 4 the immobilized biotinylatedsingle-stranded PCR product (F) can be digested with a -single-strandspecific endo- or exonuclease. For endonuclease digestion, mung beannuclease or S1 nuclease is preferred, while for exonuclease digestion,calf spleen or snake venom phosphodiesterase is used. The latter ispreferred. The immobilized digestion products (G) are purified withfurther washing in step 5, and are recovered from the solid supportusing treatment with ammonium hydroxide. As described previously, therecovered biotinylated products (G) can be analyzed after ethanolprecipitation or lyophilization.

In another variation of this embodiment (shown in FIG. 3 as "Variation2"), in step 5 an immobilized single-strand PCR product (F) (asdescribed above in step 3) is treated with ammonium hydroxide to recoverthe biotinylated single-strand PCR product (F). The ammonium hydroxideis removed using, e.g., ethanol precipitation or lyophilization and theDNA is resuspended in ultrapure water. In step 5, the isolatedsingle-stranded PCR product (F) is digested with a single-strandspecific endo- or exonuclease, preferably mung bean nuclease, S1nuclease or snake venom phosphodiesterase, respectively.

In step 6 the digestion products (G) are then analyzed, for example, viamass spectrometry or electrophoresis.

Among the advantages of the methods of the invention are: i) there is noneed to use harsh, denaturing or toxic organic compounds such as phenolor formamide, ii) biotin-streptavidin complexes can be cleaved undermild conditions, iii) volatile amines, such as ammonia, can be easilyand rapidly removed (for example by lyophilization), iv) the recoveredmaterial can be subjected to enzymatic reactions or, e.g., massspectrometric analysis, without further purification, v) a simultaneousbiotin-streptavidin complex cleavage and cation exchange. Accordingly,the process of the invention will considerably extend the applicabilityof the biotin-streptavidin system by providing a mild and selectiveprocedure to recover the biomolecules after immobilization for furtherenzymatic reactions or analysis.

Exemplification

The following examples illustrate, but do not limit, the process of thepresent invention.

Examples 1-5 illustrate the utility of the subject methods in variousapplications in diagnostics and DNA sequencing. In general, theillustrated examples involve at least some of the following steps:

1. Performing (a) an enzymatic reaction employing biotinylated DNA insolution or (b) DNA immobilized on a solid support via abiotin-streptavidin complex, respectively.

2. Separation of excess biotinylated primer, if necessary, using sizeexclusion ultrafiltration membranes.

3. Immobilization of the products of step 1a by complexing thebiotinylated DNA to a biotin-binding protein supported on a solid phase.

4. Separating the complex of step 3 or step 1b from the liquid phase.Further options include washing to remove reaction contents andimpurities, conditioning of immobilized nucleic acids, enzymaticreactions and isolation of the non-biotinylated strand of a DNA duplexfor downstream applications.

5. Treating the separated and manipulated complex of step 4 withammonium hydroxide for isolation of biotinylated molecules.

6. Complete removal of ammonium hydroxide by lyophilization orprecipitation with ethanol.

7. Downstream processing of isolated material, e.g. analysis of productsby MALDI-TOF MS or further enzymatic reactions.

As described above, the effect of ammonium hydroxide on the immobilizednucleic acids is a function of temperature and time of incubation. Ifthe immobilized material is incubated for short times at ambienttemperature, the main process is the denaturation of the immobilizeddouble-stranded molecules. However, incubation of immobilized DNA withammonium hydroxide at elevated temperatures (preferably 37-80° C.) leadsto the dissociation of the biotin-streptavidin complex.

EXAMPLE 1 MALDI-TOF MS Spectrum of a Biotinylated OligodeoxynucleotideCleaved from Streptavidin Dynabeads Using Ammonium Hydroxide

For this example, 100 pmol biotinylated oligodeoxynucleotide (20 mer),5'd(bio-AGCTCTATATCGGGAAGCCT)3' (SEQ ID NO: 1), were immobilized on 50μl streptavidin Dynabeads M-280. The beads were prepared according tothe instructions of the manufacturer. The beads were finally resuspendedin 50 μl of B/W-buffer (10 mM Tris--HCl, pH 7.5, 1 mM EDTA, 2 M NaCl).The oligodeoxynucleotide was added in a volume of 1 μl and the reactionmixture was incubated for 30 minutes at ambient temperature. Afterimmobilization, the beads were washed twice with 100 μl of ammoniumcitrate buffer (0.07 M). The beads were washed once with ultrapurewater, 25 μl of a 25% solution of ammonium hydroxide were added, and thebeads were carefully resuspended. The suspension was incubated at 60° C.for 10 minutes, and the beads were separated from the solution using amagnetic particle collector (Dynal, Hamburg, Germany). The supernatantwas saved and the procedure was repeated once. Both supernatants werecollected in a single tube. The solution was lyophilized for 30 minutes,and redissolved in 4 μl of ultrapure water. From this solution 0.5 μlwere analyzed with MALDI-TOF mass spectrometry (FIG. 4) as described inexample 2, step 4.

FIG. 4 shows a MALDI-TOF MS (15 shots, laser power 44) spectrum of abiotinylated 20 mer oligodeoxynucleotide (SEQ ID NO: 1) immobilized onstreptavidin Dynabeads and recovered using the method described above.The theoretical mass value of the biotinylated oligodeoxynucleotide is6522 Da. The mass value obtained is 6529.8 Da, in agreement with thepredicted mass. The spectrum demonstrates that a biotinylatedoligodeoxynucleotide is removed from the beads. Signals marked with anasterisk are due to depurination which occurs in the process ofionization and desorption also if ammonium hydroxide is not applied.

This Example demonstrates that biotinylated DNA can be recovered from abiotin-streptavidin complex using ammonium hydroxide at elevatedtemperatures. The oligodeoxynucleotide and the attached biotin groupremain intact and unmodified during this process. This can be concludedbecause no change of molecular weight of the original molecule wasobserved by MALDI-TOF mass spectrometric analysis after subjecting anoligodeoxynucleotide to the described procedure. Further experimentsdemonstrated that the recovered biotinylated molecule can be complexedagain to streptavidin, also suggesting an intact biotin group.

EXAMPLE 2 MALDI-TOF MS Analysis of PCR Products Purified ViaStreptavidin Dynabeads and Subsequent Removal from the Beads UsingAmmonium Hydroxide

Step

PCR was performed with 1 μl of a first set of PCR primers directedagainst the gene for the core protein of hepatitis B virus. The nestedamplification product has a length of 67 bp. 100 pmol of each primer,2.5 u Pfu (exo-) DNA polymerase (Stratagene, Heidelberg, Germany), afinal concentration of 200 μM of each dNTP and 5 μl 10× Pfu buffer (200mM Tris--HCl, pH 8.75, 100 mM KCl, 100 mM (NH₄)₂ SO₄, 20 mM MgSO₄, 1%Triton X-100, 1 mg/ml BSA, Stratagene, Heidelberg, Germany) were used ina final volume of 50 μl. The reactions were performed in a thermocycler(OmniGene, MWG-Biotech, Ebersberg, Germany) using the followingtemperature program: 94° C. for 1 minute, 60° C. for 1 minute and 72° C.for 1 minute with 20 cycles. Sequence of oligodeoxynucleotide primers(purchased HPLC-purified from MWG-Biotech, Ebersberg, Germany):

HBV13: 5'-d(TTGCCTGAGTGCAGTATGGT-)3' (SEQ ID NO:2)

HBV15bio: 5'd(bio-AGCTCTATATCGGGAAGCCT)3' (SEQ ID NO:3)

Step 2

Purification of the PCR products obtained in step 1, above, was doneaccording to the following procedure: Ultrafiltration was performedusing Ultrafree-MC filtration units (Millipore, Eschborn, Germany)according to the protocol of the manufacturer, with centrifugation at8000 rpm for 20 minutes. 25 μl (10 μl/μl) streptavidin Dynabeads M280(Dynal, Hamburg, Germany) were prepared according to the instructions ofthe manufacturer and resuspended in 25 μl of B/W buffer (10 mMTris--HCl, pH 7.5, 1 mM EDTA, 2 M NaCl). This suspension was added tothe PCR samples (still in the filtration unit) and the mixture wasincubated with gentle shaking for 15 minutes at ambient temperature. Thesuspension was transferred to a 1.5 ml Eppendorf tube and thesupernatant was separated with the aid of a magnetic particle collector,MPC, (Dynal, Hamburg, Germany). The beads were washed twice with 50 μlof 0.07 M ammonium citrate solution, pH 8.0 (the supernatant was removedeach time using the MPC).

Step 3

To remove the PCR product (a double strand of 67 base pairs), the beadswere resuspended in 25 μl of 25% NH₄ OH Suprapur (Merck, Darmstadt,Germany) and incubated at 60° C. for ten minutes. The supernatant wasremoved and saved, and the NH₄ OH treatment was repeated once. Bothsupernatants were dried in a speedvac and resuspended in 4 μl ofultrapure water (MilliQ UF plus, Millipore, Eschborn, Germany). ForMALDI-TOF MS analysis, 0.5 μl of this preparation was used.

Step 4

Half a microliter (0.5 μl) of the sample was pipetted onto the sampleholder, then immediately mixed with 0.5 μl of matrix solution (0.7 M3-hydroxypicolinic acid in 50% acetonitrile, 70 mM ammonium citrate).This mixture was dried at ambient temperature and introduced into themass spectrometer (FIG. 5). All spectra were taken in positive ion modeusing a Finnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany),equipped with a reflectron (5 keV ion source, 20 keV postacceleration)and a 337 nm nitrogen laser. Calibration was done with a mixture of anucleic acid 40 mer and a 100 mer. Each sample was measured with variouslaser energies. In the negative (control) samples, the PCR product wasnot detected at low or high laser energies. In the positive samples, thePCR product was detected at different places of the sample spot and alsowith varying laser energies.

FIG. 5 shows a MALDI-TOF MS (15 shots, laser power 48) spectrum of a PCRproduct from the hepatitis B core-antigen-coding DNA region, purifiedwith streptavidin Dynabeads and recovered from the beads using ammoniumhydroxide, as described above.

The theoretical mass value of the non-biotinylated strand is 20792.4 Da.The theoretical mass value of the biotinylated strand is 20886.9 Da. Theaverage mass of both strands is 20839.7 Da. The mass value of the signalobtained is 20812.5 Da Since double-stranded DNA molecules are denaturedduring the process of ionization and desorption, the PCR products aredetected as single-stranded molecules and the mass value obtainedrepresents the average mass of both single strands.

This Example demonstrates that MALDI-TOF MS analysis of PCR products iscompatible with the method described herein. Compared to thepurification procedures currently used, the introduced combination of astreptavidin-coated solid support, a biotinylated analyte molecule andthe recovery of the PCR product, using ammonium hydroxide, led toimproved sample processing and analysis.

EXAMPLE 3 MALDI-TOF MS Analysis of Sanger Sequencing Products PurifiedUsing Streptavidin Dynabeads and Subsequent Treatment with AmmoniumHydroxide

For the streptavidin-biotin purification, a biotinylated USP (universalsequencing primer), which was supplied HPLC-purified from PharmaciaBiotech (Freiburg, Germany), was used. The primer sequence was designedso that 10 bases downstream from the primer binding site would besequenced. The reaction was carried out using the reagents from thesequencing kit for Sequenase Version 2.0 (Amersham, Arlington Heights,Ill., USA)

Step 1

For annealing primer and template, 40 pmol bioUSP (1 μl) and 40 pmol oftemplate (1 μl) were incubated with 4 μl of Sequenase-Buffer (5×),heated to 65° C. for 2 min. and then slowly cooled down to ambienttemperature. To the annealing mixture, 1 μl Mn-buffer (supplied in thekit by the manufacturer), 1 μl dithiothreitol (DTT), 2 μl ultrapureWater (MilliQ, Millipore, Eschborn, Germany) and 2 μl diluted Sequenase2.0 (6 U, diluted with pyrophosphatase) were added.

After the addition of the Sequenase, 3 μl of the reaction mix werepipetted into each of the four termination mixes (A, C, G and T, each 4μl). The mixtures were incubated at 34° C. for 20 minutes. Each reactionwas stopped with 1.5 μl 500 mM EDTA.

Step 2

After stopping the termination reaction with EDTA, the sequencingproducts were immobilized on streptavidin beads prepared according tothe protocol of the manufacturer. To each reaction, 20 μl of prewashedbeads were added and incubated at ambient temperature for 15 minutesunder gentle shaking. After immobilization, the beads were washed twicewith B/W-buffer (see above) and once with ultrapure water. The templatewas then denatured from the immobilized strand with ultrapure water at95° C. for 2 minutes. After denaturation the beads were washed twicewith 0.07 M ammonium citrate and once with ultrapure water.

Step 3

The immobilized sequencing products were cleaved from the beads with 20μl of 25% ammonia at 60° C. for 10 minutes; the cleavage reaction wasperformed twice and the supernatants combined. The samples were thenlyophilized, resuspended in 4 μl ultrapure water and directly used forMALDI-TOF-MS.

Step 4

The reaction products were analyzed using MALDI-TOF MS (56 shots, laserpower 51) (FIG. 6) according to the procedure described in example 2,step 4.

Primer 17 mer (molecular weight 5744.4 Da):

5'd(bioGTAAAACGACGGCCAGT)3' (SEQ ID NO:4),

Template 50 mer (molecular weight 15338 Da):

5'd(TTGCGTACACACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCT C)3' (SEQ ID NO:5)

Both primer and template were synthesized (about 0.2 μmol) on a Milligen7500 using β-cyanoethylphosphoamidite chemistry (Sinha, N. D., et al.(1984) Nucleic Acids Res. 12, 4539-4577), and purified via RP-HPLC.

FIG. 6 shows the reaction of the Sanger sequencing reaction purifiedwith M-280 streptavidin Dynabeads. The sequencing reaction was carriedout in solution and the sequencing products were captured withDynabeads. The reaction contents, such as salts, enzymes, dNTPs anddideoxynucleotide triphosphates (ddNTPs), were removed by washing, andthe sequencing products were recovered for MALDI-TOF-MS analysis usingammonium hydroxide, as described above.

Three termination products were expected in the A-reaction with a lengthof 22-, 26- and 27 bases, respectively. These products are representedby the signals at 7282.6 Da, 8502.5 Da and 8809.5 Da, respectively. Thesignals at 5721.3 Da and 15325 Da represent primer and template,respectively. The signal at 9131.5 Da is due to a single-nucleotideextension of the run-through product.

This Example, and Example 4 and 5, infra, demonstrate the utility of thesubject methods in DNA sequencing. This Example shows that the method iscompatible with the analysis of conventional Sanger sequencing.

EXAMPLE 4 MALDI-TOF-MS Analysis of Exonucleolytic Digestions of PCRProducts

Step 1

The reaction mix contained 200 μM dCTP, dTTP and 200 μM C⁷ -deaza dATPand C⁷ -deaza dGTP, 100 pmol of forward and reverse primer, 100 ngM13mp18 RF, and 2.5 U of Pfu (exo-) DNA polymerase (Stratagene,Heidelberg, Germany) in a buffered solution (20 mM Tris--HCl, pH 8.75,10 mM KCl, 10 mM (NH₄)₂ SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 0.1 mg/mlBSA, Stratagene, Heidelberg, Germany) of 100 μl. The reactions wereperformed in a thermocycler (OmniGene, MWG-Biotech, Ebersberg, Germany)using the following temperature program: Initial denaturing step of 3min. 94° C., followed by 25 cycles of 94° C. for 1 minute, 48° C. for 1minute and 72° C. 1 minute.

The reverse primer was synthesized (about 0.2 μmol) on a Milligen 7500and purified with RP-HPLC. The biotinylated forward primer was purchasedHPLC-purified from Pharmacia Biotech.

Sequences:

Forward primer: 5'd(bio-GTAAAACGACGGCCAGT)3' (SEQ ID NO:6)

Reverse primer: 5'd(GAGATCTCCTAGGGGCC)3' (SEQ ID NO:7)

Step 2

The PCR product was separated from unincorporated primer byultrafiltration through a -10,000- Da molecular weight cutoff membrane.Ultrafiltration was done using Ultrafree-MC filtration units (Millipore,Eschborn, Germany) according to the protocol of the provider withcentrifugation at 8000 rpm for 20 minutes.

25 μl (10 μg/μl) streptavidin Dynabeads M-280 with a nominal size of 2.8μm (Dynal, Hamburg, Germany) were prepared according to the instructionsof the manufacturer and resuspended in 25 μl of B/W buffer (10 mMTris--HCl, pH 7.5, 1 mM EDTA, 2 M NaCl). This suspension was added tothe PCR samples (still in the filtration unit) and the mixture wasincubated with gentle shaking for 15 minutes at ambient temperature. Thesuspension was transferred to a 1.5 ml Eppendorf tube and thesupernatant was separated with the aid of a Magnetic Particle Collector,MPC, (Dynal, Hamburg, Germany). After immobilization the double-strandedPCR product was denatured using 20 μl 8 M urea. After removing the urea(containing the non-biotinylated strand) the beads were washed twicewith 50 μl of 0.07 M ammonium citrate solution, pH 8.0 (the supernatantwas removed each time using the MPC). To perform the cleavage reactionand recover the biotinylated single stranded PCR product, the beads wereresuspended in 25 μl of 25% NH₄ OH suprapur (Merck, Darmstadt, Germany)and incubated at 60° C. for ten minutes. The supernatant was removed andsaved, the NH₄ OH treatment was repeated once. Both supernatants weredried in a speedvac and resuspended in 2 μl of ultrapure water (MilliQUF plus, Millipore, Eschborn, Germany).

Step 3

1 μl of the resuspended DNA from step 2 was mixed with 0.2×10⁻³ U ofsnake venom phosphodiesterase (Boehringer Mannheim, Germany) andincubated for 20 min. at 37° C. The reaction was mixed with 1 μl matrixsolution (0.7 M 3-hydroxypicolinic acid in 50% acetonitrile, 70 mMammonium citrate) and directly used for MALDI-TOF-MS analysis.

Step 4

The reaction products were analyzed using MALDI-TOF MS (141 shots, laserpower 51) (FIG. 7) according to the procedure described above.

FIG. 7 shows a spectrum of an exonucleolytic digest of a 60 mer PCRproduct. The biotinylated PCR product was immobilized on streptavidinDynabeads and denatured with 8 M urea. The biotinylated single strandwas recovered from the beads using treatment with ammonium hydroxide.After lyophilization, the single-stranded 60 mer was digested with snakevenom phosphodiesterase. As described in example 4, the spectrumrepresents a digestion time of 20 minutes at 37° C.

Example 5 MALDI-TOF MS Analysis of an Exonuclease Digestion ofImmobilized Oligonucleotides

Step 1

400 pmol of the biotinylated 25 mer oligonucleotide were incubated with2 mg of Dynabeads according to the protocol of the provider in a finalvolume of 100 μl.

Step 2

The immobilized oligonucleotide was digested by addition of 3 μl snakevenom phosphodiesterase (6×10⁻³ U) (Boehringer Mannheim, Germany) atroom temperature.

Step 3

Aliquots of 20 μl were taken after digestion for 4, 10, 15, 20 and 25minutes and were subjected to the same purification procedure asdescribed in Example 2, step 2, supra.

Step 4

The reaction products were analyzed using MALDI-TOF MS according to theprocedure described above (FIG. 8).

For the digestion, a 5'-biotinylated oligonucleotide purchasedHPLC-purified from Biometra (Gottingen, Germany) was used.

Sequence: 5'd(bio-TACATTCCCAACCGCGTGGCACAAT)'3 (SEQ ID NO:8)

FIG. 8 shows an exonucleolytic digestion of a biotinylated 25 merimmobilized on Dynabeads. After 4 minutes of digestion with snake venomphosphodiesterase the products where purified and removed from thebeads, as described above. From this spectrum, the base sequence frombase 13 to base 25 can be seen.

Examples 4 and 5 demonstrate that the method described herein can beused for isolation of single-stranded DNA accessible for subsequentenzymatic degradation. Example 4 shows an application where formation ofa biotin-streptavidin complex, followed by treatment with ammoniumhydroxide, is used to isolate a single strand PCR product for thepurpose of digestion with a single-strand-specific exonuclease todetermine the nucleotide sequence. This experiment also demonstratesthat the recovered biotinylated material remains unmodified andaccessible for enzymatic reactions. In example 5, the digestion wascarried out while the single-stranded PCR product was still immobilizedand the products were recovered using the cleavage method introducedherein.

As can be seen from the Examples, the subject method provides a processfor purification and analysis of biotinylated molecules. The inventivemethod is compatible with mass spectrometric analysis, and with thepotential of performing further enzymatic reactions.

In another aspect, the invention provides a kit for analyzing abiotinylated biomolecule. In one embodiment, the kit includes abiotin-binding compound immobilized on a solid support, and an amine.The biotin-binding compound and amine are preferably sealed in separatecontainers. In preferred embodiments, the kit further includesinstructions for dissociating a biotin compound:biotin-binding compoundcomplex with the amine.

The contents of all reference and published patent applications citedthroughout this specification are hereby incorporated by reference intheir entirety.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 9                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 20               GCCT                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 # 20               TGGT                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 20               GCCT                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #              50GCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCTC                      - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #               25 TGGC ACAAT                                                 - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #       12                                                                    __________________________________________________________________________

We claim:
 1. A method for dissociating a biotin compound:biotin-bindingcompound complex, the method comprising:contacting the complex with aneffective amount of an amine, under conditions such that the complex isdissociated; thereby releasing a biotin compound and a biotin-bindingcompound.
 2. The method of claim 1, wherein the complex is contactedwith an amine at a temperature of between about 25° C. and about 100° C.3. The method of claim 1, wherein the biotin compound is a biotinylatedmacromolecule.
 4. The method of claim 3, wherein the biotinylatedmacromolecule is selected from the group consisting of a biotinylatednucleic acid sequence, a biotinylated protein, a biotinylatedcarbohydrate, and a biotinylated lipid.
 5. The method of claim 1,wherein the biotin-binding compound is selected from the groupconsisting of avidin and streptavidin.
 6. The method of claim 5, whereinthe biotin-binding compound is immobilized on a solid support.
 7. Themethod of claim 6, wherein the solid support is a magnetic bead.
 8. Themethod of claim 1, wherein, after the contacting step, the biotincompound is separated from the biotin-binding compound.
 9. The method ofclaim 8, wherein after the separation step, the biotin compound ispurified.
 10. The method of claim 9, wherein the biotin compound ispurified by a method selected from the group consisting oflyophilization, precipitation, filtration, and dialysis.
 11. The methodof claim 1, wherein, prior to the contacting step, the complex ispurified.
 12. The method of claim 1, wherein the biotin compound isimmobilized on a solid support.
 13. The method of claim 1, wherein,after dissociation of the complex, at least one of the biotin compoundand the biotin-binding compound is analyzed by mass spectrometry. 14.The method of claim 1, wherein, after dissociation of the complex, thebiotin-binding compound retains biotin-binding activity.
 15. The methodof claim 1, wherein, after dissociation of the complex, the biotinmoiety of the biotin compound remains substantially intact.
 16. Themethod of claim 1, wherein the amine is ammonia.
 17. The method of claim1, wherein the amine is a primary amine.
 18. A method for analyzing abiotinylated nucleic acid, comprising:contacting the biotinylatednucleic acid with a biotin-binding compound, thereby forming abiotinylated nucleic acid:biotin-binding compound complex; contactingsaid complex with an effective amount of an amine, under conditions suchthat the complex is dissociated; thereby releasing a biotinylatednucleic acid and a biotin-binding compound; and analyzing thebiotinylated nucleic acid.
 19. The method of claim 18, wherein thenucleic acid is DNA.
 20. The method of claim 18, wherein thebiotin-binding compound is immobilized on a solid support.
 21. Themethod of claim 18, wherein the biotinylated nucleic acid is analyzed bymass spectrometry.
 22. The method of claim 18, wherein the amine isammonia.
 23. The method of claim 18, wherein, after the first contactingstep, the biotinylated nucleic acid is subjected to an enzymaticreaction.
 24. The method of claim 23, wherein the enzymatic reactioncomprises treatment with an enzyme selected from the group consisting ofendonucleases and exonucleases.
 25. The method of claim 6, wherein,prior to the contacting step, the complex is purified.
 26. The method ofclaim 18, wherein, after the second contacting step, the biotinylatednucleic acid is subjected to an enzymatic reaction.
 27. The method ofclaim 18, wherein, before the first contacting step, the biotinylatednucleic acid is subjected to an enzymatic reaction.
 28. The method ofclaim 26, wherein the enzymatic reaction comprises treatment with anenzyme selected from the group consisting of endonucleases andexonucleases.
 29. The method of claim 27, wherein the enzymatic reactioncomprises treatment with an enzyme selected from the group consisting ofendonucleases and exonucleases.
 30. The method of claim 20, wherein,after the first contacting step, the biotinylated nucleic acid issubjected to an enzymatic reaction.
 31. The method of claim 20, wherein,after the second contacting step, the biotinylated nucleic acid issubjected to an enzymatic reaction.
 32. The method of claim 20, wherein,before the first contacting step, the biotinylated nucleic acid issubjected to an enzymatic reaction.
 33. The method of claim 1, whereinthe complex is contacted with an amine at a pH of between about 7 andabout
 14. 34. The method of claim 1, wherein the complex is contactedwith an amine at a concentration of between about 5% and about 28%. 35.The method of claim 1, wherein:the amine has the formula NR'₃ or N⁺ R'₄,in which each R' is independently selected from hydrogen, alkyl,cycloalkyl, alkenyl, alkynyl and aryl.
 36. The method of claim 18,wherein:the amine has the formula NR'₃ or N⁺ R'₄, in which each R' isindependently selected from hydrogen, alkyl, cycloalkyl, alkenyl,alkynyl and aryl.
 37. The method of claim 30, wherein the enzymaticreaction comprises treatment with an enzyme selected from the groupconsisting of endonucleases and exonucleases.
 38. The method of claim31, wherein the enzymatic reaction comprises treatment with an enzymeselected from the group consisting of endonucleases and exonucleases.39. The method of claim 32, wherein the enzymatic reaction comprisestreatment with an enzyme selected from the group consisting ofendonucleases and exonucleases.